Estimates of Soil Bacterial Ribosome Content and Diversity Are Significantly Affected by the Nucleic Acid Extraction Method Employed

Modern sequencing technologies allow high-resolution analyses of total and potentially active soil microbial communities based on their DNA and RNA, respectively. In the present study, quantitative PCR and 454 pyrosequencing were used to evaluate the effects of different extraction methods on the ab...

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Bibliographic Details
Published in:Applied and environmental microbiology 2016-05, Vol.82 (9), p.2595-2607
Main Authors: Wüst, Pia K, Nacke, Heiko, Kaiser, Kristin, Marhan, Sven, Sikorski, Johannes, Kandeler, Ellen, Daniel, Rolf, Overmann, Jörg
Format: Article
Language:English
Subjects:
Actinobacteria
Bacteria
Bacteria - chemistry
Bacteria - genetics
Bacteriological Techniques - methods
Biodiversity
Cells
Deoxyribonucleic acid
DNA
DNA, Bacterial - genetics
DNA, Bacterial - isolation & purification
Genes
Methods
Nucleic Acids - chemistry
Nucleic Acids - genetics
Nucleic Acids - isolation & purification
Phylogeny
Polymerase chain reaction
Proteobacteria
Real-Time Polymerase Chain Reaction - methods
Ribonucleic acid
Ribosomes - chemistry
Ribosomes - genetics
RNA
RNA, Bacterial - genetics
RNA, Bacterial - isolation & purification
RNA, Ribosomal, 16S - genetics
RNA, Ribosomal, 16S - isolation & purification
Sequence Analysis, DNA
Soil - chemistry
Soil Microbiology
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Summary:Modern sequencing technologies allow high-resolution analyses of total and potentially active soil microbial communities based on their DNA and RNA, respectively. In the present study, quantitative PCR and 454 pyrosequencing were used to evaluate the effects of different extraction methods on the abundance and diversity of 16S rRNA genes and transcripts recovered from three different types of soils (leptosol, stagnosol, and gleysol). The quality and yield of nucleic acids varied considerably with respect to both the applied extraction method and the analyzed type of soil. The bacterial ribosome content (calculated as the ratio of 16S rRNA transcripts to 16S rRNA genes) can serve as an indicator of the potential activity of bacterial cells and differed by 2 orders of magnitude between nucleic acid extracts obtained by the various extraction methods. Depending on the extraction method, the relative abundances of dominant soil taxa, in particular Actino bacteria and Proteobacteria, varied by a factor of up to 10. Through this systematic approach, the present study allows guidelines to be deduced for the selection of the appropriate extraction protocol according to the specific soil properties, the nucleic acid of interest, and the target organisms.
ISSN:0099-2240
1098-5336
DOI:10.1128/AEM.00019-16